EPR spectroscopic and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. With these methods, we have shown that Cu,Zn-superoxide dismutase (Cu,Zn-SOD), in addition to its dismutase activity, can also catalyze the generation of free radicals with hydrogen peroxide as substrate. We revealed that some Cu,Zn-SOD mutants, e.g. G93A and A4V, associated with familial amyotrophic lateral sclerosis (FALS), exhibit a gain-of-function in their ability to catalyze free radical generation, while their dismutase activity remains unchanged. The enhanced free radical generating capacity is due to the decrease in the Km value for hydrogen peroxide for the mutant relative to the wild type enzyme. To verify this correlation, we are currently overexpressing another mutant, G37R, for a similar study. In addition, it has been suggested that FALS Cu,Zn-SOD exhibits higher activity for catalyzing peroxynitrite-mediated tyrosine nitration relative to the wild type enzyme. However, we are unable to confirm this report when compared with human wild type enzyme. Nitric oxide reacts with superoxide anion to form peroxynitrite under oxidative stress. We have shown that peroxynitrite-mediated tyrosine nitration of the protein tyrosine kinase phosphorylation site cannot be phosphorylated. To further investigate the effect of tyrosine nitration on its regulatory function, we studied the binding capacity of two src homology 2 (SH2) recognition peptides, ENAEYLDLDC and CGDNDYIIPL, to the SH2 domain at the N-terminal and the C-terminal of the phospholipase C type gamma (PLC-gamma), respectively. Using the BIAcore system, we found that the phosphorylated ENAEYLDLDC and CGDNDYIIPL bind to the N-terminal and C-terminal with a Kd of 0.22 microM and 0.33 microM, respectively. However, the nitrated and the unphosphorylated peptides fail to bind to the PLC-gamma. This suggests that the irreversible tyrosine nitration will permanently impair the regulatory function of cyclic cascades involving tyrosine phosphorylation. Protein tyrosine phosphorylation plays a key role in signal transduction. In addition to tyrosine kinase, it is believed that protein tyrosine phosphatase (PTP), which exhibits its function via the formation of a thiophosphate intermediate on its active site cysteine, is participating in mediating cell signaling when reactive oxygen species are involved. We are investigating the oxidative stress-mediated inactivation of PTP-1beta. Using our home-built electroporator system, we are exploring the possibility of developing a rapid kinetic method taking advantage of the fact that reactants can be separated by loaded vesicles, which can be ruptured very rapidly by electric field pulses. However, one needs to overcome the heterogeneity problems due to vesicle preparation and vesicle rupture.